{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T09:26:37Z","timestamp":1790760397432,"version":"4.1.0"},"reference-count":62,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":36,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:00:00Z","timestamp":1782864000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["acsess.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J of Env Quality"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Freeze\/thaw pretreatment is commonly applied to animal slurry samples in laboratory studies, but its impact on organic matter transformation and methane (CH\n                    <jats:sub>4<\/jats:sub>\n                    ) emissions remains uncertain. In this study, slurry incubations were performed at 20\u00b0C with three treatments differing in freshness of feces, urine, and inoculum. CH\n                    <jats:sub>4<\/jats:sub>\n                    production and metabolite dynamics were monitored over 30 days. Freeze\/thaw of feces and urine combined with fresh inoculum yielded the highest and most stable CH\n                    <jats:sub>4<\/jats:sub>\n                    production (20\u00a0g kg\n                    <jats:sup>\u22121<\/jats:sup>\n                    volatile solids), supported by the gradual transformation of protein\u2010derived metabolites and no inhibition. In contrast, fully fresh material led to rapid hydrolysis and volatile fatty acids accumulation and slight inhibition of methanogenesis, whereas freezing the inoculum caused a pronounced delay likely due to impairment of methanogens. Metabolomics identified protein\u2010related compounds (e.g., L\u2010phenylalanine, 5\u2010aminovaleric acid, and L\u2010norleucine) as early substrates correlated to CH\n                    <jats:sub>4<\/jats:sub>\n                    formation. In contrast, lignin\/carboxyl\u2010rich aliphatic molecules and lipid compounds persisted as residual fractions. These results demonstrate that inoculum freshness is the decisive factor controlling freeze\/thaw effects on CH\n                    <jats:sub>4<\/jats:sub>\n                    production and should be prioritized when preparing substrates for laboratory incubations. When fresh materials are not practical, freeze\/thaw feces and urine can substitute it when combined with fresh inoculum.\n                  <\/jats:p>","DOI":"10.1002\/jeq2.70235","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T04:53:51Z","timestamp":1786078431000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Influence of substrate freshness on metabolite dynamics and methane emissions from pig slurry during laboratory incubation"],"prefix":"10.1002","volume":"55","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-0151-1128","authenticated-orcid":false,"given":"Rui","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Biological and Chemical Engineering Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9929-674X","authenticated-orcid":false,"given":"Michael J\u00f8rgen","family":"Hansen","sequence":"additional","affiliation":[{"name":"Department of Biological and Chemical Engineering Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0955-0327","authenticated-orcid":false,"given":"Sasha Daniel","family":"Hafner","sequence":"additional","affiliation":[{"name":"Department of Biological and Chemical Engineering Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2437-064X","authenticated-orcid":false,"given":"Frederik Rask","family":"Dalby","sequence":"additional","affiliation":[{"name":"Department of Biological and Chemical Engineering Aarhus University  Aarhus Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,6]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-024-76392-z"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0960-8524(97)84352-0"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1039\/C7EW00049A"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jproteome.0c00251"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1023\/A:1014080526608"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chemosphere.2021.131309"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.watres.2019.115199"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.watres.2022.119240"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jclepro.2021.128106"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/jeq2.20512"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1002\/jeq2.20603"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0252881"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.est.2c08891"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.est.0c01231"},{"key":"e_1_2_11_16_1","unstructured":"Dalby F. R. Wang R. &Hansen M. J.(2024 September).Potential contribution of feed spillage in pig barns to slurry methane emission.Proceedings of International symposium on gas and dust emissions from livestock Universitat Polit\u00e8cnica de Val\u00e8ncia Valencia Spain."},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/2016GL071930"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aeaoa.2024.100258"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/es3018489"},{"key":"e_1_2_11_20_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-015-6623-0"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2021.126337"},{"key":"e_1_2_11_22_1","doi-asserted-by":"publisher","DOI":"10.3390\/su14169934"},{"key":"e_1_2_11_23_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.mi.39.100185.001251"},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.3389\/fenvs.2019.00178"},{"key":"e_1_2_11_25_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0323024"},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jclepro.2022.130772"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2019.121518"},{"key":"e_1_2_11_28_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssuschemeng.8b01601"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.watres.2022.119488"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10532-021-09935-0"},{"key":"e_1_2_11_31_1","doi-asserted-by":"publisher","DOI":"10.3390\/w11112420"},{"key":"e_1_2_11_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.rser.2023.113804"},{"key":"e_1_2_11_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2009.12.125"},{"key":"e_1_2_11_34_1","doi-asserted-by":"publisher","DOI":"10.2134\/jeq2007.0386"},{"key":"e_1_2_11_35_1","unstructured":"Nielsen O.\u2010K. Plejdrup M. S. Winther M. Nielsen M. Gyldenk\u00e6rne S. Mikkelsen M. H. Albrektsen R. Hjelgaard K. H. Fauser P. &Bruun H. G.(2025).Denmark's National Inventory Document 2025: Emission Inventories 1990\u20132023\u2014Submitted under the United Nations Framework Convention on Climate Change and the Paris Agreement(Report No. 655).Aarhus University DCE \u2010 Danish Centre for Environment and Energy."},{"key":"e_1_2_11_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cej.2025.165971"},{"key":"e_1_2_11_37_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jenvman.2024.120233"},{"key":"e_1_2_11_38_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0160968"},{"key":"e_1_2_11_39_1","unstructured":"Picarro. (2021).G4301 GasScouter datasheet.Picarro Inc.https:\/\/www.picarro.com\/sites\/default\/files\/product_documents\/Picarro_G4301%20GasScouter%20Datasheet_211101_0.pdf"},{"key":"e_1_2_11_40_1","doi-asserted-by":"publisher","DOI":"10.2175\/106143005X86655"},{"key":"e_1_2_11_41_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.wasman.2026.115659"},{"key":"e_1_2_11_42_1","unstructured":"Regeringen. (2024).Aftale om et Gr\u00f8nt Danmark.https:\/\/regeringen.dk\/aktuelt\/tidligere\u2010publikationer\/aftale\u2010om\u2010et\u2010groent\u2010danmark\/"},{"key":"e_1_2_11_43_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.180.4.767-772.1998"},{"key":"e_1_2_11_44_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.energy.2017.07.146"},{"issue":"6","key":"e_1_2_11_45_1","first-page":"460","article-title":"Glucose fermentation to acetate, CO2 and H2 in the anaerobic hyperthermophilic eubacterium Thermotoga maritima: Involvement of the Embden\u2010Meyerhof pathway","volume":"161","author":"Schr\u00f6der C.","year":"1994","journal-title":"Archives of Microbiology"},{"key":"e_1_2_11_46_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2860(00)00724-9"},{"key":"e_1_2_11_47_1","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms11071825"},{"key":"e_1_2_11_48_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2020.122869"},{"key":"e_1_2_11_49_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac202661j"},{"key":"e_1_2_11_50_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2016.07.031"},{"key":"e_1_2_11_51_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1005399107"},{"key":"e_1_2_11_52_1","doi-asserted-by":"publisher","DOI":"10.2134\/jeq2015.09.0446"},{"key":"e_1_2_11_53_1","doi-asserted-by":"publisher","DOI":"10.1002\/jeq2.70186"},{"key":"e_1_2_11_54_1","doi-asserted-by":"publisher","DOI":"10.3168\/jds.S0022-0302(91)78551-2"},{"key":"e_1_2_11_55_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jenvman.2022.115319"},{"key":"e_1_2_11_56_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chemosphere.2022.134117"},{"key":"e_1_2_11_57_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chemosphere.2024.141157"},{"key":"e_1_2_11_58_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.watres.2012.03.021"},{"key":"e_1_2_11_59_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.scitotenv.2019.03.175"},{"key":"e_1_2_11_60_1","doi-asserted-by":"publisher","DOI":"10.3390\/su16073052"},{"key":"e_1_2_11_61_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.watres.2020.115783"},{"key":"e_1_2_11_62_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2018.05.049"},{"key":"e_1_2_11_63_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biortech.2019.122237"}],"container-title":["Journal of Environmental Quality"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/acsess.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jeq2.70235","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/acsess.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/jeq2.70235","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/acsess.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jeq2.70235","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T09:58:13Z","timestamp":1787651893000},"score":1,"resource":{"primary":{"URL":"https:\/\/acsess.onlinelibrary.wiley.com\/doi\/10.1002\/jeq2.70235"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7]]},"references-count":62,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2026,7]]}},"alternative-id":["10.1002\/jeq2.70235"],"URL":"https:\/\/doi.org\/10.1002\/jeq2.70235","archive":["Portico"],"relation":{},"ISSN":["0047-2425","1537-2537"],"issn-type":[{"value":"0047-2425","type":"print"},{"value":"1537-2537","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7]]},"assertion":[{"value":"2026-01-18","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-07-12","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-06","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e70235"}}